Combinations of quinoxaline-di-n-oxides and tetracycline antibiotics



United States Patent O US. Cl. 424-227 8 Claims ABSTRACT OF THEDISCLOSURE Chemical reduction of water-soluble quinoxaline-di-N- oxidesin aqueous solution by zinc metal is inhibited by contacting the zincmetal with an aqueous solution of a water soluble tetracyclineantibiotic to form a zinc complex of tetracycline antibiotic coatingthereon. The quinoxaline-di-N-oxides, which are antibacterial agents,may be administered to animals and poultry via drinking water, withwater-soluble tetracycline antibiotic as co-solute to inhibit reductionby zinc metal surface of commonly used galvanized iron drinking troughs.Compositions comprising S-hydroxytetracycline and certainquinoxaline-di- N-oxides such as2-methyl-3-hydroxymethylquinoxalinedi-N-oxide, and2-formylquinoxaline-di-N-oxide and Schitf bases thereof, exhibit asynergistic effect in the control of chronic respiratory disease ofpoultry and in promoting the growth of animals, and also inhibit invitro drug resistance development of E. coli.

Cross references to related applications This application is acontinuation-in-part of copending applications Ser. Nos. 468,153, filedJune 29, 1965 which is now US. Patent No. 3,344,022, 470,934, filed July9, 1965 which is now US. Patent No. 3,371,090 and 587,420, filed Oct.18, 1966. These applications in turn are, respectively,continuations-in-part of Ser. Nos. 393,- 311, filed Aug. 31, 1964,397,504, filed Sept. 16, 1964, and 502,602, filed Oct. 22, 1965, all ofwhich are abandoned.

This application discloses subject matter relating to the invention ofcopending application of Conover and Johnston, entitledTetracycline-induced Inhibition of Photochemical Decomposition ofQuinoXaline-N,N'-dioxides.

Background of the invention This invention relates to a method ofinhibiting the chemical reduction of water-soluble quinoxaline-di-N-oxides in aqueous solution by zinc metal, and in particular to the useof tetracycline antibiotics to inhibit said chemical reduction. Thisinvention also relates to novel compositions comprising a tetracyclineantibiotic and quinoxaline-di-N-oxide, and more particularly tocompositions comprising S-hydroxytetracycline and aquinoxaline-di-N-oxide which are especially effective for the control ofchronic respiratory disease in poultry and as growth promotants fordomestic animals and to animal feeds containing said compositions.

It is known that many quinoxaline-di-oxides are useful as antibacterialagents, but heretofore their use as chemotherapeutic agents has beenimpaired to a certain extent by the ease with which these compounds arechemically reduced. For example, attempts to administerquinoxaline-di-N-oxides to poultry via drinking water are often impairedsince the zinc metal surface of the commonly used galvanized irontroughs and trays reduces the comice pounds to products which do notshare the antibacterial activity of the quinoxaline-di-N-oxides.

The term chronic respiratory disease as used throughout this applicationincludes complicated chronic respiratory disease, infections sinusitisand related diseases. The field condition commonly known as air sacdisease, air-sacculitis or complicated chronic respiratory disease isgenerally considered to involve Mycoplasma and secondary infections,especially those caused by coliforms such as Escherichia coli, Proteusand Micrococcus species. Mycoplasma, especially M. gallinarum, M,gallisepticum, M. iners, M. synovaei and type N-PPLO(pleuropneu-monia-like organisms) are believed to be the primary agentsresponsible for chronic respiratory and related diseases of poultry andof infectious sinusitis of turkeys.

The economic significance of such avian diseases has led to extensiveresearch on practical measures of immunization and effective therapeuticproducts. Various antibiotics, including S-hydroxytetracycline, havebeen used at levels of to 500 gms./ton of feed or in the animalsdrinking water at concentration of 0.5 to 1.0 gm./ gal. with somebenefit, probably by their control of susceptible secondary bacterialinfections. Intra-muscular injection of streptomycin andchlortetracycline is also reported to be beneficial. Price et al.,Poultry Science 36, 219-225 (1957) found 55-hydroxytetracycline at 100and 500 gms./ ton of feed markedly reduced coliform invasion in chronicrespiratory disease infected chickens and had some influence on thecourse of the disease. The increased beneficial effect observed at thehigher level was attributed to direct action of the drug against theetiological agent. Recent studies by Sojka et al. (Res. Vet. Sci. 2,340, 1961) have shown a surprising increase in the incidence of E. colistrains resistant to antibiotics. Because of this, the countermeasuresdeveloped for the control of chronic respiratory disease have not beentotally satisfactory.

The development of quinoxaline-di-N-oxides as agents especiallyeffective for the control of chronic respiratory disease is described byJohnston in US. Ser. Nos. 468,153, filed June 29, 1965, 470,934, filedJuly 9, 1965, and 587,420, filed Oct. 18, 1966. These compounds controlnot only the secondary infections associated with chronic respiratorydisease but are surprisingly effective against Mycoplasma and promotegrowth and feed efficiency in both diseased and healthy poultry and indomestic animals in general. Stephens U.S. Patent 2,974,167, issued Mar.7, 1961, discloses that the 1:1 adducts of the tetracycline antibiotics(tetracycline, 5-oxytetracycline and 7- chlorotetracycline) with2-methyl-3-formylquinoxalinedi-N-oxide exhibit biological activityagainst a variety of pathogenic microorganisms. The instant inventiondiffers from the Stephens invention in that the latter relates todistinct chemical compounds which are necessarily formed in asubstantially non-aqueous inert organic solvent. These 1:1 adducts arenot present in compositions of the instant invention and are not formedin carrying out the methods of the instant invention.

Summary of the invention It has been unexpectedly found that reductionof quinoxaline-di-N-oxides in aqueous solution by zinc metal can beinhibited by coating the surface of the zinc metal with a zinc complexof a tetracycline antibiotic. By tetracycline antibiotic is meantthroughout that class of compounds possessing a common type ofstructure, that is, each of these compounds has a hydronaphthacene ringsystem, highly substituted with oxygen-containing groups and othersubstituents; this class of compounds is meant to include compoundsnaturally produced by fermentation processes, compounds produced bytotal synthesis and derivatives of those compounds produced by furtherchemical transformation. Also included in this class are the varioussalts of aforesaid tetracycline antibiotics. The coating can be easilyapplied to the surface by contacting the zinc metal surface with anaqueous solution of a water-soluble tetracycline antibiotic; the use ofa watersoluble salt of tetracycline antibiotic is most convenient.Effective inhibition is achieved either by pretreating the zinc metalsurface with the tetracycline antibiotic solution or by simply adding aquantity of the tetracycline antibiotic to the aqueous solution ofquinoxaline-di-N- oxide.

This method of inhibiting reduction of quinoxaline-di- N-oxides inaqueous solution by zinc metal is both extremely effective andconvenient. Since tetracycline antibiotics are often administered toanimals and poultry via drinking water, it is now highly desirable tocombine the administration of these compounds with that of thequinoxaline-di-N-oxides. The use of a single solution to administer bothantibacterial agents not only simplifies the treatment of the animalsand poultry, but also unexpectedly serves to stabilize thequinoxaline-di-N-oxides against chemical reduction. Although it might beex pected that certain compounds other than the tetracycline antibioticswill inhibit the reduction of quinoxaline-di-N- oxides, it is animportant and unexpected advantage of the instant invention that thiscondition can be achieved by compounds having substantialchemotherapeutic activity themselves.

The method of the instant invention protects the N-oxide function of allwater-soluble quinoxaline-di-N- oxides in aqueous solution againstreduction by zinc metal. However, a preferred embodiment of thisinvention is the protection of those quinoxaline-di-N-oxides especiallynoted for their antibacterial or growth promoting properties, viz,water-soluble quinoxaline-di-N-oxides of the general formulae wherein Ris selected from the group consisting of hydrogen, alkyl of up to carbonatoms, ot-hydroxy lower alkyl, a-lower alkanoyloxy lower alkyl anda-lower alkoxy lower alkyl; R is selected from the group consisting ofakyl of up to 10 carbon atoms, lower alkanoyl, u-hydroxy lower alkyl,a-lower alkanyloxy lower alkyl and u-lower alkoxy lower alkyl; R and Rtaken together, are selected from the group consisting of trimethylene,tetramethylene and 1,4-dihydroxytetramethylene; and X X X and X are eachselected from the group consisting of hydrogen and lower alkyl.

A particularly preferred embodiment of the instant invention iscompositions or mixtures comprising S-hydroxytetracycline and aquinoxaline-di-N-oxide selected from the group consisting of thosehaving the formulae wherein R is selected from the group consisting ofmethyl, hy-

droxy-methyl, acetoxyrnethyl and forrnyl;

R is selected from the group consisting of hydro-gen,

methyl, hydroxymethyl and acetoxymethyl:

With the proviso that R; is hydrogen when R is Formyl;

R and R when taken together are -CH(OH)CH CH(OH) R is selected from thegroup consisting of ureido (-NHCONH thionoureido (-NHCSNH2); carbo(lower alkoxy)amino (-NHCOOR wherein the lower alkoxy group 0R containsup to four carbon atoms; 3-(2-oxazolidonyl) hydroxy; methylamino; andacetylamino; and

R is selected from the group consisting of hydrogen and methyl, since ithas now been found that these compositions exhibit a synergistic effectin the control (therapy and propylaxis) of chronic respiratory diseaseof poultry and in promoting the growth of domestic animals such aspoultry (chickens, turkeys, ducks), lambs, cattle, swine, goats, horses,dogs and cats.

The use of various antibiotics for the treatment of chronic respiratorydisease and for accelerating the growth of domestic animals has longbeen known. However, the combination of S-hydroxytetracycline and aquinoxaline-di-N-oxide such as those enumerated above results in asynergistic effect in the treatment of chronic respiratory disease andan accelerated rate of growth wholly unexpected from that heretoforeobserved.

Detailed description of the invention An especially preferred embodimentin the method of inhibition of chemical reduction is the class ofwatersoluble quinoxaline-di-N-oxides which possess usefulchemotherapeutic properties, such as2-methyl-3-hydroxymethylquinoxaline-di-N-oxide,2,3-dimethylquinoxalinedi-N-oxide, 2-methylquinoxaline-di-N-oxide,1,2,3,4-tetrahydrophenazine-di-N-oxide,1,4-dihydroxy-1,2,3,4-tetrahydrophenazine-di-N-oxide and2,3-trimethylenequinoxaline-di-N-oxide. Most preferred is2-methyl-3-hydroxymethylquinoxaline-di-N-oxide. These compounds arehighly effective in maintaining weight gain and feed consumption ofpoultry in the presence of chronic respiratory disease and incontrolling chronic respiratory disease, and in accelerating growth andimproving feed efiiciency of animals.

Inasmuch as the inhibition of reduction of the preferred class ofquinoxaline-di-N-oxides is usually of concern when administering thecompounds to animals and poultry, it is desirable to use a tetracyclineantibiotic which would be similarly administered for its ownchemotherapeutic value. For example, tetracycline, 5-hydroxytetracycline, 7-chlorotetracycline,6-deoxy-6-demethyl-6-methylene-S-hydroxytetracycline (hereinafterreferred to as methacycline) and a-6-deoxy5-hydroxytetracycline(hereinafter referred to as doxycycline) are illustrative oftetracycline antibiotics which have this advantage.S-hydroxytetracycline is a preferred tetracycline antibiotic. Of course,it is understood that tetracycline anti biotics with other substitucntswould be equivalent for the purpose of the instant invention, even ifthey would not normally be administered for their own chemotherapeuticproperties.

A preferred method for coating the zinc metal surface is to contact thesurface with an aqueous solution of a water-soluble tetracyclineantibiotic. For example, exposure of the zinc metal surface to aqueousS-hydroxytetracycline hydrochloride results in the formation of a yellowstain on the metal surface. This stain, the Zinc complex ofS-hydroxytetracycline, appears to be much like a mono layer, which canbe removed from the zinc metal surface by means such as scraping onlywith great difiiculty. Inasmuch as the complex coating forms ratherquickly, it is not necessary to pretreat the surface to obtainprotection of the quinoxaline-di-N-oxides; the S-hydroxytetracyclinehydrochloride may be present in the quinoxaline-di-N-oxide solution as aco-solute and reduction of the latter compound is still effectivelyinhibited.

If the coating is to be formed by contacting the surface with aqueoussolution of the tetracycline antibiotic, a water-soluble salt of thetetracycline antibiotic is preferably used since the water-soluble saltsare soluble in water to a much greater degree than is the amphotericform of the antibiotics. Inasmuch as the degree of protec tion of thequinoxaline-di-N-oxides is a function of the amount of tetracycline insolution, it is usually much more convenient to use the water-solublesalts; said salts will often permit the use of various tetracyclineantibiotics the amphoteric forms of which are insufficiently soluble inwater to result in an effective degree of protection. Of course, to theextent that the amphoteric antibiotics may be sufliciently soluble inwater to permit effective and convenient coating of the metal surface,it is understood that the use of the amphoteric form is completelyequivalent to the use of the water-soluble salts.

If the tetracycline antibiotic is to be a co-solute and be administeredalong with the quinoxaline-di-N-oxide, there is no problem of thetetracycline antibiotic being present in an insuflicient amount to beeffective in inhibiting reduction of quinoxaline-di-N-oxide; an amountsufiicient for therapeutic purposes will be sufficient to protect thequinoxaline-di-N-oxide. It is preferable that between about a 0.10 and10.0 molar equivalent amount of tetracycline antibiotic be used withrespect to the amount of quinoxaline-dFN-oxide used. However, it isunderstood that the use of tetracycline antibiotics in any amount willbe effective to a certain extent; insofar as any zinc complex forms acoating on the surface of the metal, a corresponding degree ofinhibition will result. Between about 0.4 and 8.0 molar equivalentamount of tetracycline antibiotic is more preferred, with about 1.2molar equivalent amount being most advantageous. If the tetracyclineantibiotic is to be a co-solute and be administered along with thequinoXaline-di-N-oxide, the water-soluble salt must be pharmaceuticallyacceptable. By pharmaceutically acceptable is meant those saltformingacids and metals which do not substantially increase the toxicity of theamphoteric antimicrobial agent.

The pharmaceutically acceptable acid addition salts include salts ofmineral acids such as hydrochloric hydroiodic, hydrobromic, phosphoric,metaphosphoric, nitric and sulfuric acids, as well as salts of organicacids such as tartaric, acetic, citric, malic, benzoic, glycollic,gluconic, gulonic, succinic, arylsulfonic, e.g. p-toluenesulfonic acids,and the like. The pharmaceutically acceptable metals include sodium,potassium and alkaline earth metals of atomic number up to and including20, i.e. magnesium and calcium, and additionally, aluminum, zinc, ironand manganese, among others. Of course, the metal salts include complexsalts, i.e. metal chelates, which are well recognized in thetetracycline art.

The pharmaceutically unacceptable salts of the tetracycline antibioticsmay be used if the zinc metal surface is pretreated to form a coating ofthe zinc complex, with the surface then washed clean of pharmaceuticallyunacceptable species prior to the use of the quinoxaline-di- N-oxidesolution. The pharmaceutically unacceptable acid addition salts includethose formed with hydrofluoric and perchloric acids. Thepharmaceutically unacceptable metal salts embrace most commonly salts oflithium and of alkaline earth metals of atomic number greater than 2'0,i.e. barium and strontium.

Inasmuch as the instant invention relates to a method of stabilizingquinoxaline-di-N-oxides against chemical reduction, it is noted that theinvention of aforesaid copending application of Conover and Johnstondoes not make the instant invention obvious to one Skilled in the art,and similarly, is not itself made obvious in light of the instantinvention. The phenomenon of chemical reduction by zinc metal is notrelated to, or comparable to, photochemical decomposition; a method ofpreventing either of the two phenomena would not suggest to one skilledin the art that the other could be prevented in a similar fashion.

When the combinations of 5-hydroxytetracycline andquinoxaline-di-N-oxides are used in animal feeds, theS-hydroxytetracycline component is used to the extent of from about 10to about 2000 g. per ton of feed and the quinoxaline-di-N-oxides to theextent of from about 10 to about 500 g. per ton of feed.

The effectiveness of the herein described combinations against coliforminfections is considered to be one of synergism of the effect of thequinoXaline-di-N-oxide by the S-hydroxytetracycline since the coliformtest infections are substantially resistant to S-hydroxytetracycline.

For a given level of S-hydroxytetracycline, the addition of one of theherein described quinoxaline-di-N- oxides increases the activity of theS-hydroxytetracycline against resistant E. coli to such a significantextent as to be considered synergistic. Insofar as the combinations ofS-hydroxytetracycline and quinoxaline-di-N-oxides appear to inhibit thein vitro drug resistance development of E. coli, it is expected thatthese combinations might produce a similar retardation of in vivoresistance development. This phenomenon would offer a possible way ofreducing quinoxaline-di-N-oxide toxicity by permitting a reduction indosage. Likewise, the addition of S-hydroxytetracycline to a given levelof one of said quinoxaline-di-N-oxides produces a synergistic effect inthe activity of the quinoxaline-di-N-oxide against PPLO infections.

In the novel compositions or mixtures of this invention the ratio ofantibiotic to quinoxaline-di-N-oxide is from about 1:50 to about 200:1.The preferred ratio of antibiotic to quinoxaline-di-N-oxide is fromabout 1:1 to about 20:1. The quantity of novel composition administereddepends, of course, upon the route of administration and the purpose forwhich the composition is used, e.g. control of chronic respiratorydisease, growth promotion, and the animal to be treated.

For the purposes described herein the novel and valuable compositions ofthis invention can be administered orally or parenterally. Subcutaneousand intramuscular injections are the preferred methods of parenteralinjection for several reasons; simplicity, convenience and the compoundsappear less toxic. According to one modification of the presentinvention the compositions described herein when used therapeuticallyare administered orally or parenterally, e.g. by subcutaneous orintramuscular injection, so as to provide each component of thecomposition to poultry in a dosage of from about 1 mg./kg. to aboutmg./kg. of body weight for the control of chronic respiratory disease.When administered orally it is preferred to use a dosage which provideseach component at from about 1 mg./kg. to about 60 mg./kg. of bodyweight. For parenteral administration dosages which provide eachcomponent at from about 10 mg./kg. to about 100 mg./kg. of body weightare preferred.

For prophylactic use these novel compositions are administered so as toprovide from about 10 to about 100 rug/kg. of body weight of aquinoxaline-di-N-oxide and from about 10 to about 500 mg./kg. ofantibiotic daily.

These compositions can be used either in the form of solutions orsuspensions, aqueous or non-aqueous. When administered parenterally, asingle dose is generally sufficient, but in the event multiple doses areemployed, the dosage is repeated at a suitable interval, e.g. weekly,monthly. Vehicles suitable for parenteral injection may be eitheraqueous such as water, isotonic saline, isotonic dextrose, Ringerssolution, or non-aqueous such as fatty oils of vegetable origin (cottonseed, peanut oil, corn, sesame), dimethylsulfoxide and other non-aqueousvehicles which will not interfere with the therapeutic efficiency of thepreparation and are non-toxic in the volume or proportion used(glycerol, propylene glycol, sorbitol). Additionally, compositionssuitable for extemporaneous preparation of solutions prior toadministration may advantageously be made. Such compositions may includeliquid diluents, for example, propylene glycol, diethyl carbonate,glycerol, sorbitol, etc.; buffering agents, as well as hyaluronidase(spreading factor), local anesthetics and inorganic salts to afforddesirable pharmacological properties.

In general no tissue irritation results from the subcutaneous injectionof these compositions. However, as a precaution against possibleirritation and to facilitate absorption, parenteral administration ofthese compositions in combination with hyaluronidase can be employed. Anincrease in the rate of absorption of the drugs is observed and theoccasional discomfort on injection is greatly reduced, if not completelyeliminated. Hyaluronidase levels of at least about 150 (U.S.P.) unitsare very effective in this respect. Higher or lower levels can, ofcourse, be used but 150 units per dose appears to give consistently goodresults as evidenced by the absence of edema and the general behavior ofthe poultry following injection of the drug preparation.

Dry mixtures containing the active ingredients together with salt(sodium chloride) and/or buffering agents or local anesthetics areprepared for extemporaneous use. A total concentration of activeingredients in such mixtures of at least about 50% is useful.

According to a further modification of the present invention, thesevaluable compositions are administered to poultry by the oral route byincorporating said compositions in the daily rations so as to provideeach component at a dosage of from about 1 to about 60 mg./kg. of bodyweight. This can be achieved by a number of methods including mixingwith a nutritionally-balanced feed, the preparation of concentrates,dosage unit formulations such as capsules, tablets, liquid mixtures andsolutions, or they can be administered in admixture with minerals suchas sodium chloride which are frequently fed to poultry as a supplement.Dilute solutions or suspensions, e.g. a 0.1% solution, can be suppliedfor drinking purposes. Indeed, levels of about 300500 mg./gal. ofquinoxaline-di-N- oxide, e.g. 2 methyl-3-hydroxymethylquinoxaline-di-N-oxide, plus 1000 mg./ga1. of a salt of tetracycline antibiotic, e.g.S-hydroxytetracycline, have been found very effective in the treatmentof chronic respiratory disease. Of course, the actual concentration tobe used will be determined by the person skilled in the art whoprescribes the use of these combinations.

The unique feed compositions of this invention are found to beparticularly valuable for use with poultry and especially for poultryinfected with chronic respiratory disease. A type of conventional feedmaterial which may be employed is recommended to contain roughly between50% and 80% of grains, between and 10% animal protein, between 5% and30% vegetable protein, between 2% and 4% minerals together withsupplemental vitaminaceous sources. When a feed containing a majorproportion of these substances and a minor proportion of one of theherein described compositions is employed, the poultry shows a markedimprovement, if not complete recovery, over the infection and reach thedesirable weight in a shorter period of time than usual with a markedlygreater feed efficiency. It should be noted that these valuable productseliminate, or at least minimize, the economic losses normally associatedwith chronic respiratory discase.

It has further been found that the addition of a low level of the hereindescribed antibiotic-quinoxaline-di-N- oxide compositions to the diet ofanimals, both ruminant and non-ruminant, such that these animals receivethe product over an extended period of time, so as to provide eachcomponent at a level of from about 1 mg./kg. to about 100 mg./kg. ofbody weight per day, especially over a major portion of their activegrowth period, re-

sults in an acceleration of the rate of growth and improved feedefiiciency. Included in these two classes of animals are poultry(chickens, ducks, turkeys), cattle, sheep, dogs, cats, swine, rats,mice, horses, goats, mules, rabbits, mink, etc. The beneficial effectsin growth rate and feed efiiciency are over and above what is normallyobtained with complete nutritious diets containing all the nutrients,vitamins, minerals, individual components of the herein describedcompositions and other factors known to be required for the maximumhealthy growth of such animals. The animals thus attain market sizesooner and on less feed.

The herein described feed compositions have been found to beparticularly valuable and outstanding in promoting the growth of suchanimals as poultry, rats, hogs, swine, lambs, cattle, and the like. Insome instances the degree of response may vary with respect to the sexof the animals. The products may, of course, be administered in onecomponent of the feed or they may be blended uniformly throughout amixed feed; alternatively as noted above, they may be administered in anequivalent amount via the animals water ration. It should be noted thata variety of feed components may be of use in the nutritionally balancedfeeds.

The resulting new feed compositions have marked effects on the rate ofgrowth and feed efficiency (the number of pounds of feed required toproduce a pound gain in weight). The novel feed supplements of thisinvention permit the use of higher energy, higher protein diets toobtain improved feed/ gain ratios and the use of feedstuffs that atpresent are not utilized efi'iciently. Simply stated, the compositionsof this invention when fed to animals are more efficiently converted toanimal body weight than prior art compositions. Any animal feedcomposition may be prepared to comprise the usual nutritional balance ofenergy, proteins, minerals, and vitamins together with one or more ofthe antibiotic-quinoxalinedi-N-oxide combinations described herein. Someof the Various components are commonly grains such as ground grain, andgrain by-products; animal protein substances, such as meat, and fishby-products; vitaminaceous mixtures, e.g. vitamin A and D mixtures,riboflavin supplements and other vitamin B complexes; and bone meal,limestone, and other inorganic compounds to provide minerals.

The relative proportions of the antibiotic and quinoxaline-di-N-oxide infeeds and feed concentrates may vary somewhat, depending upon thecompound, the feed with which they are employed and the animal consumingthe same. These substances are advantageously combined in such relativeproportions with edible carriers to provide concentrates which mayreadily be blended with standard nutritionally balanced feeds or whichmay be used themselves as an adjunct to the normal feedings.

Dry pre-mixes containing these compounds are prepared.containing from0.10 to about 10% of the active ingredients mixed with salt (sodiumchloride) and other minerals which it is desired to incorporate into thepoultry ration. This can then be fed on an ad libitum basis by adjustingthe proportion of active ingredient in the mixture to the average dailyconsumption per animal so as to provide the proper daily dose asspecified above. If prepared feed supplements are employed, the materialcan be administered in admixture with the feed. Again a concentrationrange of about 0.10 to 10% of the drugs in the feed is employed.However, higher proportions can be satisfactorily employed dependingupon the palatability of the product to the animal. This can be readilydetermined by simple experimentation. It is sometimes convenient to mixthe daily dose with only a portion of the average daily allotment toinsure complete consumption of the dose. The balance of the daily feedsupplement can then be fed after consumption of the medicated portion inthe usual fashion. These methods are particularly useful forprophylactic treatment, but

similar compositions can be employed for therapeutic use.

In the preparation of concentrates a wide variety of carriers may beemployed containing the aforesaid drugs. Suitable carriers include thefollowing: soybean oil meal, corn gluten meal, cotton seed oil meal,sunflower seed meal, linseed oil meal, cornmeal, limestone and corncobmeal. The carrier facilitates uniform distribution of the activematerials in the finished weed with which the concentrate is blended.This is especially important because only a small proportion of thesepotent materials are required. The concentrate may be surface coated, ifdesired, with various proteinaceous materials or edible waxes, such aszein, gelatin, microcrystalline wax and the like to provide a protectivefilm which seals in the active ingredients. It will be appreciated thatthe proportions of the drugs in such concentrates are capable of widevariation since the amount of active materials in the finished feed maybe adjusted by blending the appropriate proportion of concentrate withthe feed to obtain the desired degree of supplementation. In thepreparation of high potency concentrates, i.e. premixes, suitable forblending by feed manufactures to produce finished feeds or concentratesof lower potency, the content of each drug component may range fromabout 0.1 g. to 50 g. per pound of concentrate. A particularly usefulconcentrate is provided by blending 2 g. of drug composition with 1pound of limestone or 1 pound of limestone soybean oil means (1:1).Other dietary supplements, such as vitamins, minerals, etc. may be addedto the concentrates in the appropriate circumstances.

The concentrates described may also be added to animal feeds to producea nutritionally balanced, finished feed containing from about 10 g. toabout 500 g. of the quinoxaline-di-N-oxide and from about 10 g. to about2000 g. of S-hydroxytetracycline per ton of finished feed. In the caseof ruminants the finished feed should contain protein, fat, fiber,carbohydrate, vitamins and minerals, each in an amount sufficient tomeet the nutritional requirements of the animal for which the feed isintended. Most of these substances are present in naturally ocurringfeed materials, such as alfalfa hay or meal, cracked corn, Whole oats,soybean oil meal, corn silage, ground corn cobs, wheat bran, and driedmolasses. Bone meal, limestone, iodized salt and trace minerals arefrequently added to supply the necessary minerals, and urea to provideadditional nitrogen.

As is well known to those skilled in the art, the types of diets areextremely variable depending upon the purpose, type of feedingoperation, species, etc. Specific diets for various purposes are listedby Morrison in the Appendix of Feeds and Feeding, the MorrisonPublishing Company, Clinton, Iowa, 1959.

In the case of non-ruminant animals, such as hogs, a suitable feed maycontain from about 50 to 80% of grains, 3 to 10% animal protein, 5 to30% vegetable protein, 2 to 4% of minerals, together with supplementaryvitaminaceous sources.

The following examples illustrate in greater detail the manner ofpracticing the present invention. They are, however, not to be construedas limiting the scope thereof in any way. All values reported areaverage values. The efficacy index is defined as 100 times the ratio ofthe pounds of feed per pound of grain produced by the unsupplementedfeed to that produced by the supplemented feed.

EXAMPLE I The following solutions were prepared and immediatelyintroduced into galvanized iron cans (6" height, 4" diameter):

Can 12-methyl-3-hydroxymethylquinoxaline-di-i -oxide (50 mg, 0.25 mmole)in one liter of water Can22-methyl-3-hydroxymethylquinoxaline-di-N-oxide (50 mg., 0.25 mmole) andS-hydroxytetracycline hydrochloride (150 mg., 0.30 mmole) in one literof water Can 35-hydroxytetracycline hydrochloride (150 mg.,

0.30 mmole) in one liter of Water.

The amount of 2-methyl-3-hydroxymethylquinoxaline-di- N-oxide in Cans 1and 2 was determined by ultraviolet spectroscopy at various timeintervals. Water was used as a blank for the solution of Can 1 andsolution from Can 3 was used as a blank for the solution of Can 2.

Percent qulnoxaliue-di-N- oxide Can 1 Can 2 Hour After 168 hours ofexposure to the galvanized iron surface, the solution of Can 2 wasanalyzed by polarography and found to contain 100% of the originalamount of 2-methyl-3-hydroxymethylquinoxaline-di-N-oxide.

EXAMPLE II EXAMPLE III The procedure of Example I is repated whereinequivalent amounts of the following compounds are used, individually, inplace of said S-hydroxytetracycline hydrochloride, with the rate ofreduction of 2-methyl-3- hydroxymethylquinoxaline-di-N-oxide in Can 2being substantially less than that in Can 1 in each instance:

tetracycline hydrochloride 7-chlorotetracycline hydrochloridemethacycline hydrochloride doxycycline hydrochloride amphoterictetracycline amphoteric-S-hydroxytetracyclihe EXAMPLE IV The procedureof Example I is repeated wherein equiva lent amounts of the followingquinoxaline-di-N-oxides are used, individually, in place of said2-methyl-3-hydromethylquinoxaline-di-N-oxide, with the rate of reductionof the quinoxaline-di-N-oxide in Can 2 being substantially less thanthat in Can 1 in each instance:

2,3-dimethylquinoxaline-di-N-oxide Z-methylquinoxaline-di-N-oxide 1,2,3,4-tetrahydrophenazine-di-N-oxide 1,4-dihydroxy- 1 ,2,3,4-tetrahydrophenazine-di-N-oxide 2,3-trimethylenequinoxaline-di-N-oxideEXAMPLE V One liter of aqueous S-hydroxytetracycline hydrochloridem-g./l.) is placed in a galvanized iron can (6 height, 4" diameter) for5 days, after which time the solution is poured out and replaced withone liter of aqueous 2 methyl-3 hydroxymethylquinoxalinedi-N-oxide (50mg./ 1.). After 48 hours of exposure to the galvanized iron surface, thelatter solution is analyzed by ultraviolet spectroscopy and found tocontain 98% of the original amount of Z-methyl 3hydroxymethylquinoxaline-di-N-oxide, compared to 51% when the can wasnot pretreated with S-hydroxytetracycline hydrochloride.

1 1 EXAMPLE v1 EXAMPLE VII The process of Example V is repeated whereinequivalent amounts of the following compounds are used, individually, inplace of said S-hydroxytetracycline hydrochloride:

tetracycline hydrochloride 7-chlorotetracycline hydrochloridemethacycline hydrochloride doxycycline hydrochloride amphoterictetracycline amphoteric S-hydroxytetracycline trolling coliforminfection in chickens is demonstrated as follows.

Four-week old chickens are infected with coliform air sacculitis byinjecting 0.5 ml. of a 24-hour broth culture of twoS-hydroxytetracycline resistant, avian pathogenic isolates of E. colistandardized to 47% light transmission into the left posterior thoracicair sec.

Seven groups of 10 birds each are used as indicated below. The treatedbirds are administered the drugs by subcutaneous inoculation into theupper cervical region at the time of exposure to E. coli. Each lot isreplicated twice. After five experimental days, all surviving chickensare sacrificed and the extent of air sac lesion determined according tothe following method of scoring: Ono visible lesion; 1lesion confined toone air sac; 2lesion involving 2 air sacs with no inflammatory extensionto adjacent tissues; 3lesion involving 3 or 4 air sacs with noinflammatory extension to adjacent tissues; 4lesion involving 4 or moreair sacs with or without inflammatory extension to adjacent tissues. Theresults, presented in Table 1, demonstrate a synergistic action againstthe coliform air sacculitis infection for the two drugs in combinationand an enhanced effect on growth.

TABLE I Quinoxaline-di- 5-hydroxy Feed N-oxide tetraconsumption dose,eyeline, Lesion Percent per lot post Wt. gain, Eflieacy mg./bird mgJbirdscore mortality injection g.[bird index 0 0 2, 916 17s. 7 100 3. 15 5 1,492 57. 3 0 5. 0 1. 5 0 160. 8 61.0 5. 0 25. 0 1. 0 2, 817 171. 4 64. 210. 0 1. 40 0 2, 826 160. 5 61. 2O 10. 0 25. 0 0. 90 O 2, 728 180. 1 88.70 25. 0 3. 05 0 1, 763 51. 6 8.60

1 Non-infected, nonmedieated controls. 2 Infected, non-medicatedcontrols.

In each instance, the rate of reduction of 2-methyl-3-hy- EXAMPLE Xdroxymethylquinoxaline-di-N-oxide is substantially less in a pretreatedcan than in a can which has not been pretreated with thetetracycline-type antibiotic.

EXAMPLE VIII The process of Example V is repeated wherein equivalentamounts of the following compounds are used, in-

TABLE II Quinoxa- 1ine-di- 5-hydroxy Feed N-oxide tetraconsumption dosecycline, Lesion Percent per lot post Wt. gain, Efficacy Lot mgJbirdmg./bird score mortality injection gJbird index 1 N on-infected,nonmedicated controls. 2 Infected, non-medicated controls.

dividually, in place of said 2-rnethyl-3-hydroxymethyl- EXAMPLE XIquinoxaline-di-N-oxide EXAMPLE TX The effectiveness of2,3-dimethylquinoxaline-di-N- oxide in combination withS-hydroxytetracycline in con- Four-week-old turkeys are infected withcoliform air sacculitis by injecting 0.5 ml. of a 24-hour broth cultureof two avian pathogenic S-hydroxytetracycline resistant isolates of E.coli (standardized to 47% light transmission) into the left posteriorthoraci air sac. The drugs are administered in the feed, the medicatedrations being fed for a five-day period starting one day prior to E.coli exposure. The feed is tendered to the birds for imbibition adlibitum. The birds, 15 per lot, are checked as to mortality, averageweight change, feed consumption and, after sacrifice 5 dayspost-injection, for air sac lesion score. The effectiveness of thecombination of 2,3-dimethylquinoxaline di N oxide and 5hydroxytetracycline is evident from the data below (Table III). Thesynergistic. ac-

13 tion of the two drugs is thus demonstrated. Also evident is asignificant improvement in weight gain.

lowing modifications. A challenge of 0.5 ml. of media containing tenmillion avian pathogenic,

TABLE III Quinoxa- Feed line-di- 5-liydr0xy consumption N -oxidetetraper lot dose, cyeline, Lesion Percent post Wt. gain, Efficacy Lotg./ton g./ton score mortality injection g./bird index l Non-infected,non-medicated control. 2 Infected, non-medicated control.

EXAMPLE XII The therapeutic value of 2 methyl3-hydroxymethylquinoxaline-di-N-oxide for the control of coliform airsacculitis in chickens is demonstrated as follows. Five lots offour-week-old chickens, 10 birds per lot, are exposed to E. coli byinjecting into the left posterior thoracic air sac 0.5 ml. of a 24-hourculture of two avian pathogenic 5- hydroxytetracycline resistanceisolates of E. coli, standardized to 47% light transmission. Eachinfected bird is administered the drug by a single subcutaneous innoculation into the upper cervical region at the time of exposure to E. coli.A lot of ten birds is used as control. Each lot is replicated twice.Pertinent data, presented in Table IV, demonstrate the synergisticaction of this drug when used in combination with S-hydroxytetracycline.

EXAMPLE XIV The efficacy of 1,4 dihydroxy 1,2,3,4 tetrahydrcphenazine diN oxide in combination with S-hydroxytetracycline in controlling PPLOinfections in chicks is demonstrated as follows. Seven lots oftwo-Week-old chickens (10 birds per lot) are exposed to PPLO infectionby injecting into the left posterior thoracic air sac 0.25 ml. of a72-hour broth culture of PPL'O. Each infected bird is then administeredthe drug by subcutaneous inoculation into the upper cervical region atthe time of exposure to PPLO. A single inoculation is administered perbird. An additional lot of ten birds serves as non-infected,nonmedicated controls. Each lot is replicated twice. The treatmentschedule and results are summarized in Table VI.

TABLE IV Quinoxalinefi hydroxy di-N-oxide tetracycline Perform- Mg./ M./kg. Mg./ Lesion Percent anee Efficacy Lot bird liW. bird ]3.W. scoremortality index+ index 100 100 10.9 0 81.8 57.7 91.0 91.5 11.7 0

Performance Indexzratio of weight of the chickens to feed efiiciency.

Non-infected. non-medicated control.

2 Infected, non-medicated control.

'1 L EXAMPLE x111 AB E Q 1 fi-hydirotxy- 6O uinoxa inee ra- Thetherapeutlcefiicacy of several qurnoxalme-di-N- dmmi-de cycnpe' LesionPercept Emcacy oxides in combination with S-hydroxytetracyclme 1S dem-Lot d s mg-l d g-l score ta ty ind x onstrated by the followingexperiments. 0 0 100 A. Four-Week-old chickens are exposed to an avian 2g 3 g pathogenic, broad spectrum antibiotic resistant E. coli 0.20 094.43 infection by injection into their left posterior thoracic air 3 81%;

sac of 0.5 ml. of media containing approximately eight million organismper ml. The test chickens are administered the drug in their feed foralive-day period beginning 48 hours prior to challenge with E. coli.Each bird received 25 million viable E. coli organisms. The lesion scoreis determined 12 days post-inoculation upon sacrifice of the chickens.Twenty birds are used per treatment.

Pertinent data are presented below in Table V-A.

B. The above procedure is repeated but with the fol 1 Non-infected,non-medicated controls. 2 Infected, non-medicated controls.

EXAMPLE XV 15 VII. Four lots of four-week-old chickens (10 birds perlot) are exposed to PPLO infection by injecting into the left posteriorthoracic air sac 0.5 ml. of a 72-hour broth culture of PPLO. Onebroad-spectrum antibiotic resistant E. coli organisms is used.Medication is begun 24 hours tetracycline in treating a PPLO infectionin four-weekold chickens is demonstrated as follows. Six lots of birds,10 birds per lot (each replicated twice), are exposed to PPLO infectionby injection, into their left posterior thoracic air sac, of 0.5 ml. ofa 72-hour broth culture of prior to challenge With E. coli and all birdsare ulthanized PPLO. The drugs are administered in the feed starting oneand autopsied ten days post-inoculation. Ten birds are day prior to PPLOexposure and continued for a 5-day used per treatment, each of which isreplicated six times. period. Unsupplemented feed is provided the birdsPertinent data is presented in Table V-B. throughout the remainder ofthe experiment. One lot of TABLE V-A 5-hydroxytetra- AveragePerformcycline, Percent Lesion ance Efficacy Quinoxaline-di-N-oxideGJton g./ton mortality score index index Non-infected, non-medicatedcontrol 0 0 100 100 Infected, non-medicated control 1 1. 65 87.7 02-formyl 200 500 0 0. 90 93.5 70. 4 2-methyl-3-acetoxymethyl 200 500 00. 50 106. 4 94. 7 2,3-diacetoxymethyl 250 500 0 0.10 106.1 103.12-Iormylsemithiocarbazone 200 500 0 0. 80 81. 8 66. 22,3-dihydroxymethyl 200 500 0 0. 103. 9 101; 0 2-methyl-3-iormylcarbethoxyhydrazone 200 500 1 0.85 96.3 69.0 2,3-dimethyl 200 500 o 0.101. s 95. 7

TABLE V-B 5-hydroxytetra- Average Performcycline, Percent Lesion anceEfficacy Quinoxaline-di-N-oxide G./ton g./ton mortality score indexindex Non-infected, nonmedicated control 0 0 100 100 Infected,non-medicated control 3 1. 25 75. 9 0 2-methyl-3-hydroxymethyl 200 500 00. 55 85. 6 64. 1 2-methyl3-acetoxymethyl 200 500 0 0. 68 77. 8 48. 82,3-diacetoxy methyl 200 500 3 0. 72 90. 2 56. 9

of these four groups serves as infected, non-medicated 10 birds,replicated twice, serves as non-infected concontrol. The remaining threegroups are administered the trol. All birds are euthanized and autopsiedon the tenth drug in their feed for a five-day period starting one dayday. All birds are cultured for PPLO at the time of prior to PPLOexposure. Upon completion of the fivesacrifice. day period the birds aresacrificed and examined for their Results are presented .in Table VIII.It will be noted air sac lesion score. One lot of ten birds serves asnonthat 2,3-dimethylquinoxaline-di-N-oXide at 125 g./ton and infected,non-medicated controls. 250 g./ton in combination withS-hydroxytetracycline at TABLE VII S-hydroxytetra- Dose, cycline, LesionPercent Efllcacy Lot Quinoxaline-di-N-oxide g./ton gJton score mortalityindex Noninfected, nontreated controls 0 0 100 Infected, non-treatedcontrols- 2. 0 0 1,4-dil1ydroxy THP+ 250 500 1.35 0 48. 10 Isomer ofl,4dihydroxy THP+ 250 500 1. 05 0 79. 2,3-dimethyl 250 500 0.35 0 93.86

THP 1,2,3,4-tetrahydrophenazine.

EXAMPLE XVI 50 The exceptional therapeutic activity of 2,3-dimethyl- 500g./ton is especially elfective,

indeed synergistic, in

quinoxaline-di-N-oxide in combination with S-hydroxycontrolling PPLO.

TABLE VIII Quinoxaline-di-N- 5-hydroxy-tetraoxide cycline Percent PPLOPPLO Lesion Efficacy Perform- Gm./ton Mg./b1rd Gm./ton Mg./bn-dmortality positive negative score index ance 1 Non-infected,non-medicated control. Infected, non-medicated control.

1 7 EXAMPLE XVII This experiment demonstrates the therapeutic activityof 2 methyl 3-hydroxymethylquinoxaline-di-N-oxide in combination with5-hydroxytetracycline against M. gallisepticum infection in chickens.

18 EXAMPLE XIX The procedure of Example XIII A is repeated using thefollowing quinoxaline-di-N-oxides in place of those of Example XIII A:

Three lots of 4 /2 week-old Cobb strain broilers, 0 birds per lot (threereplicates) are exposed to M. galliit septicwm by injecting into theleft posterior thoracic air sac 0.5 ml. of a 72-hour agar broth cultureof M. gallisepw 3 ticum. The medicated rations are provided for fivedays 10 R starting 24 hours prior to challenge with M. gallisepticum. Afourth lot of 10 birds (three replicates) serves as control. The birdsare sacrificed five days post challenge. 0 Pertinent data are presentedin Table IX.

TABLE IX Quinoxao-hydroxy line-ditetra- N-oxide, eycline, Percent LesionPeriorm- Efficacy Lot g./ton g./t0n mortality score ance index 0 0.0 100100 0 2.26 82 0 0 1. 33 100 81 o 0.70 107 86 1 Non-infected,non-medicated control. 2 Intected, non-medicated control.

EXAMPLE XVIII R One hundred four-'week-old broiler chickens are usedNHCO NHZ H to conduct this experiment designed to investigate the CH8eificacy of 2,3-dirnethylquinoxaline-di-N-oxide and S-hy- NH-COOCH: Hdroxytetracycline against a PPLO infection. On the first -NHCOOCH; CH:experimental day, all chicks are exposed to an isolate of m 000 E M.gallisepticum PPLO by injecting 0.5 ml. of a 72-hour H broth cultureinto the left posterior air sac. Two replicate pens of ten chickens eachare assigned to the following l treatments: (1) non-infected,non-medicated control; (2) H infected, non-medicated control; (3) 25mg./chick of 5- i hydroxytetracycline; (4) l0 mg./chick of 2,3-dimethyloquinoXaline-di-N-oxide; (5) 25 mg./chick of S-hydroxy- 4% CHtetracycline and 10 mg./ chick of 2,3-dimethylquinoxaline- I di-N-oxide.The foregoing drugs are administered by sub- 5 cutaneous inoculationinto the upper cervical region at 2 the time of exposure to PPLO. Afterfive experimental -OH H days all surviving chickens are sacrificed andthe extent -0H CH; of air sac lesion determined. NHGHa H The reductionin air sac lesion score observed for the NHGH3 CH administration of thedrugs in combination is greater than H S the sum of the reductlons 1nlesion score noted for the CH CH administration of these two drugsindividually (Table X). a

EXAMPLE XX TABLE X A daily transfer study in Brain heart infusion wascon- %if,i" .Zggfigig ducted with E. coli to compare the minimuminhibitory concentration (MIC) of 2-rnethyl-3-hydroxymethylquin- Lot .3%BW 3333: 2 333 oxaline-di-N-oxide, S-hydroxytetracycline, andcombinations thereof. The combinations tested had quinoxaline- 2 2 "3di-N-oxide:S-hydroxytetracycline weight ratios of 1:1, 8 g 2 1:2 and1:4. The inhibiting agent was added in increasing 0 46:32 concentrationto a series of E. coli containing tubes. The Nonintected non medicatedcontrol inoculum for each succeeding transfer was taken from the tube ofthe previous days test with highest concentra- Z Infected, non-medicatedcontrol.

19 tion of inhibiting agent which still showed growth. Thus, theinoculum came from the tube with concentration just below the MIC. Thematerial was diluted 1 10 to serve as inoculum. In the following table,QDO refers to 2-methyl-3-hydroxymethylquinoxaline-di-N-oxide and HTCrefers to S-hydroxytetracycline.

dihydroxy 1,2,3,4-tetrahydrophenazine-di-N-oxide and2,3-trimethylenequinoxaline-di-N-oxide.

5. The method of claim 1 wherein said tetracycline antibiotic is awater-soluble salt of a compound selected from the group consisting oftetracycline, S-hydroxytetracycline, 7-chlor0tetracycline,6-deoxy-6-demethyl-6- TABLE XI Inhibiting concentrations, meg/1111., ateach transfer as single Inhibiting concentration, mcgJmL, at eachtransfer entities of QDO plus H'IO combinations Transfer QDO HTC1:1/QDO/HTC 1 :2/QD O/HTC 1 :4/QDO/HTC Original MIC 62. 5 1.9 1. 9/1. 90.9/1. 9 0.4/1.9 1 250 3.9 1.9/1.9 0.9/1. 9 0.4/1.9 3. 9 3. 9/3. 9 1.9/3. 9 0.9/3.9

The above data indicate that the 1:1 combination is the most effectiveof three combinations studied and is more effective than either compoundused alone with respect to retardation of drug resistance development.

What is claimed is:

1. A method of inhibiting chemical reduction of a water-solublequinoxaline-di-N-oxide in aqueous solution by zinc metal which comprisesdissolving with said quinoxaline-di-N-oxide in said solution awater-soluble salt of a tetracycline antibiotic in an amount sufiicientto inhibit said reduction.

2. The method of claim 1 wherein said water-soluble salt of atetracycline antibiotic is pharmaceutically acceptable and the molarequivalent ratio of quinoxaline-di- N-oxideztetracycline salt is between1:01 and 1:l0.0.

3. The method of claim 1 wherein said tetracycline antibiotic is awater-soluble salt of a compound selected from the grou consisting oftetracycline, 5-hydroxytetraeycline, 7 chlorotetracycline, 6 deoxy 6demethyl-6- methylene 5 hydroxytetracycline anda-6-de0xy-5hydroxytetracycline and said water-soluble quinoxaline-di-N-oxide is selected from the group consisting of 2-methyl- 3hydroxymethylquinoxaline di N-oxide, 2,3-dimethylquinoxaline-di-N-oxide, 2 methylquinoxaline-di-N-oxide, 1,2,3,4tetrahydrophenazine-di-N-oxide, 1,4-dihydroxy-1,2,3,4-tetrahydrophenazine-di-N-oxide and2,3-trimethylenequinoxaline-di-N-oxide.

4. The method of claim 1 wherein said water-solublequinoxaline-di-N-oxide is selected from the group consisting of2-methyl-3-hydroxymethylquinoxaline-di-N-oxide,2,3-dimethylquinoxaline-di-N-oxide, 2-methylquinoxaline- :li-N-oxide,1,2,3,4-tetrahydrophenazine-di-N-oxide, 1,4-

methylene 5 hydroxytetracycline and a-6-dCOXY-S-hydroxytetracycline.

6. The' method of claim 1 wherein said tetracycline antibiotic is5-hydroxytetracycline hydrochloride.

7. A method of inhibiting chemical reduction of 2-methyl-3-hydroxymethylquinoxaline-di-N-oxide in aqueous solution by zincmetal which comprises dissolving with said quinoxaline-di-N-oxide insaid solution about 1.2 molar equivalent amount of a pharmaceuticallyacceptable water-soluble salt of a compound selected from the groupconsisting of tetracycline, S-hydroxytetracycline, 7-chlorotetracycline, 6 deoxy 6 demethyl-6-methylene-5-hydroxytetracycline and a 6 deoxy S-hydroxytetracycline.

8. The method of claim 7 wherein said salt is S-hydroxytetracyclinehydrochloride.

References Cited UNITED STATES PATENTS 2,891,062 6/1959 Ursprung 260-2672,974,167 3/1961 Stephens 260-559 3,344,022 9/1967 Johnston 167-53.13,359,160 12/1967 Gordon 167-53 3,371,090 2/1968 Johnston 260-2403,022,218 1/1962 Sherman 167-53 ALBERT T. MEYERS, Primary Examiner J. V.COSTIGAN, Assistant Examiner U.S. Cl. X.R. 424-250 UNITED STATES PATENTOFFICE CERTIFICATE OF CORRECTION Patent No. 3,497,594 February 24, 1970James D. Johnston It is certified that error appears in the aboveidentified patent and that said Letters Patent are hereby corrected asshown below:

' Column 1, line 43, "311" should read 371 and "397,504" should read397,004 Column 15, line 4, the subject matter beginning with "broad" andextending through line 33 of column 15, including both Tables V-A andV-B should be read as though it followed "pathogenic" at column 14, line2.

Signed and sealed this 15th day of September 1970.

(SEAL) Attest:

Edward M. Fletcher, Jr.

Attesting Officer WILLIAM E. SCHUYLER, IR.

Commissioner of Patents

